The formation of articular cartilage requires a complex interplay between cartilage producing cells, growth factors, and matrix macromolecules, including type II procollagen. Chondrogenesis is characterized by the production of an alternatively-spliced form of type II procollagen (IIA) containing a cysteine-rich (CR) domain in the amino-terminal (NH2) propeptide. This exon 2-encoded domain binds to BMP-2 and is homologous to motifs found in multiple copies in other secreted proteins that bind BMPs (e.g., chordin and sog). These BMP-binding proteins play critical roles in cell type determination and morphogenesis. We will test the hypothesis that the spatial presentation of CR domains in type IIA procollagen is critical for interactions with growth factors. Studies of the IIA NH2-propeptide have been hampered by the complexity of isolating collagen propeptides from tissues. We, therefore, produced a chimeric protein consisting of the IIA NH2-propeptide linked to a C00H-terminal C-type lectin trimerization cassette to mimic the presentation of the IIA CR domains in native procollagen. Accordingly, we will use this construct and mutant constructs thereof for three aims: We will: 1) determine whether the exon 2 domain alone is sufficient for optimal BMP-2 binding or whether the full-length propeptide is necessary for the correct spatial arrangement of these exon 2 domains; 2) compare the growth factor-binding activity of wild-type and mutant IIA NH2-propeptides that differ in the number and distribution of exon 2 domains; and 3) examine the function of these constructs in a biological system in vitro. Structural elucidation of wild-type propeptides will allow us to create proteins with mutations in the CR domain to decipher important amino acids involved in growth factor binding. The structure of these molecules will be characterized using biochemical and immunological techniques, and their in vitro interactions with BMP-2 will be studied using binding assays. For the third aim, we will use a highly reproducible assay that measures the BMP-dependent elongation (differentiation) of lens epithelial cells to monitor the biological activity of wild-type or mutant exon 2-containing propeptides. Together, the proposed studies will provide important data about the functional roles of exon 2 sequences in cartilage development and repair. The data obtained will assist with the development of strategies to examine the effect of over-expressing selected mutants in transgenic mice.